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n-3 Fatty acids, inflammation and immunity: new mechanisms to explain old actions

Published online by Cambridge University Press:  14 May 2013

Philip C. Calder
Philip C. Calder*
Affiliation:
Human Development and Health Academic Unit, Faculty of Medicine, University of Southampton, Tremona Road, Southampton SO16 6YD, UK
*
Corresponding author: Professor P. C. Calder, fax +44 2380 795255, email pcc@soton.ac.uk
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Abstract

Numerous effects of n-3 fatty acids EPA and DHA on functional responses of cells involved in inflammation and immunity have been described. Fatty acid-induced modifications in membrane order and in the availability of substrates for eicosanoid synthesis are long-standing mechanisms that are considered important in explaining the effects observed. More recently, effects on signal transduction pathways and on gene expression profiles have been identified. Over the last 10 years or so, significant advances in understanding the mechanisms of action of n-3 fatty acids have been made. These include the identification of new actions of lipid mediators that were already described and of novel interactions among those mediators and the description of an entirely new family of lipid mediators, resolvins and protectins that have anti-inflammatory actions and are critical to the resolution of inflammation. It is also recognised that EPA and DHA can inhibit activation of the prototypical inflammatory transcription factor NF-κB. Recent studies suggest three alternative mechanisms by which n-3 fatty acids might have this effect. Within T-cells, as well as other cells of relevance to immune and inflammatory responses, EPA and DHA act to disrupt very early events involving formation of the structures termed lipid rafts which bring together various proteins to form an effective signalling platform. In summary, recent research has identified a number of new mechanisms of action that help to explain previously identified effects of n-3 fatty acids on inflammation and immunity.

Keywords

CytokineEicosanoidFish oilResolvinLipid raft
Type
Conference on ‘Transforming the nutrition landscape in Africa’
Information
Proceedings of the Nutrition Society , Volume 72 , Issue 3 , August 2013 , pp. 326 - 336
Copyright
Copyright © The Author 2013 

Overview of inflammation and immunity in health and disease

The immune system is the means by which the sources of non-threatening antigens are identified and tolerated and by which threatening antigens are identified and their sources eliminated. The immune system is principally thought of in the context of protection against pathogenic bacteria, viruses, fungi and parasites, but it also plays roles in identification and elimination of tumour cells and in the response to physical insults such as injury, surgery, burns and irradiation. The immune system is highly complex, involving many different specialised cell types dispersed throughout the body and moving between body compartments as part of routine immune surveillance or in response to specific stimuli. The cells of the immune system interact with one another and with other cell types (e.g. epithelial cells, endothelial cells, platelets, hepatocytes and adipocytes) in order to elicit and regulate local and systemic responses to infection, injury or insult. Many chemical mediators are produced during the course of an immune response; some of these are directly damaging to infectious organisms, others play a regulatory role promoting the activity of particular cell types, while others serve to terminate the response when the source of the initial immune stimulation has been eliminated.

The immune response can be classified into two general arms termed the innate (or natural) and the acquired (or specific). The innate immune response can be activated via recognition of certain general structural features of pathogens; these features may be shared by numerous pathogens. For example, lipopolysaccharide (LPS), a component of the cell wall of Gram-negative bacteria and also known as endotoxin, is recognised by Toll-like receptor (TLR)-4 on the surface of innate immune cells. In contrast, the acquired immune response is specific for a single antigen which must be presented by an antigen-presenting cell to an antigen-specific T-cell. Thus, the innate response is induced quickly and is not improved by prior exposure to the triggering pathogen, while the acquired response is induced slowly but is enhanced by prior exposure to the antigen. The two arms of the immune system communicate during an immune response because innate immune cells can present antigen, thus inducing the acquired response, while the acquired immune response produces chemicals that activate innate immune cells or make their processes more efficient. Inflammation is part of a normal innate immune response. Obviously, immune responses, including the inflammatory component, are protective and hence are beneficial to health. However, active immune responses triggered by normally benign structures or by host antigens can cause tissue damage and disease. These diseases will often involve infiltration and activation of immune cells (both innate and acquired) within particular tissue compartments initiating and perpetuating tissue damage which can become pathological. This can be caused by inappropriate activation of the immune response, perhaps because of wrong recognition of an immune trigger (e.g. a host antigen), or by an inability to shut-off an inappropriate immune response because of loss of a terminating or resolving factor. Examples of diseases where an inappropriate immune response is central to the pathology are rheumatoid arthritis (autoimmune destruction of the joints), inflammatory bowel diseases (loss of tolerance to commensal gut bacteria resulting in an active and damaging immune response within the gastrointestinal mucosa) and asthma (adverse immune response to a normally benign environmental antigen causing airways damage). Classic inflammatory cells and chemical mediators produced by those cells are central to the pathology of these diseases and hence they are often referred to as ‘inflammatory diseases’( Reference Calder, Albers and Antoine 1 – Reference Calder, Ahluwalia and Albers 3 ). Nevertheless it should not be overlooked that cells of the acquired immune response also play an important, often key regulatory, role in these diseases.

n-3 Fatty acids, inflammation and immunity

Research on the influence of fatty acids on immunity started in the 1970s, with the earliest studies evaluating and comparing the effects of common SFA and the n-6 fatty acid linoleic acid( Reference Meade and Mertin 4 – Reference Yaqoob 7 ). The effects observed were considered to involve modifications of the physical state of the plasma membrane of immune cells: membrane order (‘fluidity’) could clearly be involved in a membrane-mediated process such as phagocytosis( Reference Mahoney, Hamill and Scott 8 – Reference Calder, Bond and Harvey 11 ) but has also been thought to be important in T-lymphocyte responses to activation( Reference Anel, Naval and González 12 , Reference Calder, Yaqoob and Harvey 13 ). The discovery that eicosanoids, including PGE2, play roles in inflammation and in the regulation of immune cell function( Reference Goldyne and Stobo 14 , Reference Kinsella, Lokesh and Broughton 15 ) initiated research into the effects of the common eicosanoid precursor arachidonic acid and also raised the possibility that the effects of some fatty acids on immune cell responses could be due to modification of eicosanoid production( Reference Kinsella, Lokesh and Broughton 15 , Reference Calder, Bevan and Newsholme 16 ). Thus, the two long-standing mechanisms to describe the effects of fatty acids on inflammation and immunity involve alterations of membrane order and modulation of eicosanoid production, both driven by modification of the fatty acid composition of the phospholipids within membranes of inflammatory and immune cells.

The first studies of the effects of n-3 fatty acids on inflammation and immunity were published in the 1980s. In vitro studies revealed that both EPA and DHA could influence the functional responses of immune cells to stimulation (see Calder( Reference Calder 5 ) for references) and early studies of fish oil in experimental models of autoimmunity( Reference Leslie, Gonnerman and Ullman 17 ) and clinical trials of fish oil in patients with rheumatoid arthritis( Reference Kremer, Bigauoette and Michalek 18 , Reference Kremer, Jubiz and Michalek 19 ) demonstrated significant anti-inflammatory activity of the combination of EPA and DHA. The effects of EPA and DHA on inflammation and immunity were considered to be consistent with the existing mechanisms of fatty acid action. First, the highly unsaturated nature of EPA and DHA means that they have the potential to have marked effects on membrane order in immune cells( Reference Calder, Yaqoob and Harvey 13 ). Secondly, incorporation of EPA and DHA into cells involved in inflammation and immunity is partly at the expense of arachidonic acid( Reference Lokesh, Hsieh and Kinsella 20 – Reference Rees, Miles and Banerjee 37 ) hence decreasing the amount of substrate available to produce inflammatory and immunoregulatory eicosanoids( Reference Endres, Ghorbani and Kelley 26 – Reference Sperling, Benincaso and Knoell 29 , Reference Meydani, Endres and Woods 38 – Reference Von Schacky, Kiefl and Jendraschak 42 ). Over the last 25 years, the effects of n-3 fatty acids on aspects of inflammation and immunity have been extensively examined. They have been demonstrated to affect the functions of a range of cell types involved in innate and acquired immunity, to modify the expression of key cell surface proteins and to modulate the production of reactive oxygen species, eicosanoids and cytokines (Table 1). The effects of n-3 fatty acids on inflammation and immunity have been reviewed many times( Reference Calder 5 , Reference Yaqoob 7 , Reference Calder 43 – Reference Shaikh, Jolly and Chapkin 54 ), and the reader is referred to these reviews for a detailed coverage of the topic. The current article will provide an update on the mechanisms of action of n-3 fatty acids with respect to inflammation and immunity, putting these in the context of the earlier understandings of n-3 fatty acid actions.

Table 1. Summary of the effects of n-3 fatty acids (EPA+DHA) on immune and inflammatory cells

EPA and DHA are rapidly incorporated into phospholipids of immune cells in human subjects

It is well known that increased oral supply of EPA and DHA results in an increase in the amount of those fatty acids in immune cells in laboratory animals( Reference Lokesh, Hsieh and Kinsella 20 – Reference Fritsche, Alexander and Cassity 24 , Reference Yaqoob and Calder 39 – Reference Peterson, Jeffery and Thies 41 ) and human subjects( Reference Endres, Ghorbani and Kelley 26 – Reference Thies, Nebe-von-Caron and Powell 33 , Reference Kew, Mesa and Tricon 35 – Reference Rees, Miles and Banerjee 37 Reference Endres, Ghorbani and Kelley 26 – Reference Thies, Nebe-von-Caron and Powell 33 , Reference Kew, Mesa and Tricon 35 – Reference Rees, Miles and Banerjee 37 , Reference Browning, Walker and Mander 55 ). This increase occurs in a dose–response manner( Reference Healy, Wallace and Miles 32 , Reference Rees, Miles and Banerjee 37 , Reference Browning, Walker and Mander 55 ) and time-course studies reported that near maximum incorporation of both EPA and DHA occurred within a few weeks in human subjects( Reference Yaqoob, Pala and Cortina-Borja 31 , Reference Rees, Miles and Banerjee 37 , Reference Browning, Walker and Mander 55 ). The incorporation of these highly unsaturated long chain n-3 fatty acids is mainly at the expense of n-6 fatty acids, especially arachidonic acid( Reference Lokesh, Hsieh and Kinsella 20 – Reference Rees, Miles and Banerjee 37 ). A recent study evaluated the incorporation of EPA and DHA into human mononuclear cells (a mixture of 85% lymphocytes and 15% monocytes isolated from peripheral blood) over one week( Reference Faber, Berkhout and Vos 56 ). By coincidence this study provided the same daily amounts of EPA and DHA as used in an earlier study by Yaqoob et al.( Reference Yaqoob, Pala and Cortina-Borja 31 ), allowing a direct comparison of the findings of these two studies in healthy human volunteers. Faber et al.( Reference Faber, Berkhout and Vos 56 ) report significant incorporation of EPA and DHA after just 1 d of supplementation and combining the findings of these two studies reveals that the near maximum incorporation of both n-3 fatty acids into human mononuclear cells occurs at about 7 d of supplementation (Fig. 1). This rapid incorporation of EPA and DHA suggests that subsequent functional effects on cell responsiveness and function may occur more quickly than previously considered.

Fig. 1. Time course of incorporation of EPA and DHA and of disappearance of arachidonic acid in human mononuclear cells in healthy volunteers consuming fish oil. Healthy human volunteers consumed fish oil providing 2·1 g EPA and 1·1 g DHA per d for 1 week( Reference Faber, Berkhout and Vos 56 ) or for 12 weeks( Reference Yaqoob, Pala and Cortina-Borja 31 ). Blood was sampled at several time points in each study and mononuclear cells prepared. Fatty acid composition of the cells was determined by GC. Mean values are shown. ▪ and , EPA; • and , DHA; ▴ and , arachidonic acid; Black symbols represent data from Faber et al.( Reference Faber, Berkhout and Vos 56 ); Grey symbols represent data from Yaqoob et al.( Reference Yaqoob, Pala and Cortina-Borja 31 ).

Opposing actions of eicosanoids produced from arachidonic acid and EPA regulate leucocyte-endothelial interaction

Eicosanoids are biologically active lipid mediators produced from PUFA, most commonly the n-6 fatty acid arachidonic acid. Eicosanoids play wide ranging roles in inflammation and regulation of immune function( Reference Lewis, Austen and Soberman 57 , Reference Tilley, Coffman and Koller 58 ). To produce these eicosanoids, arachidonic acid is released from membrane phospholipids through the action of phospholipase A2 enzymes, and then acts as a substrate for cyclooxygenase (COX), lipoxygenase or cytochrome P450 enzymes (Fig. 2). COX enzymes lead to PG and thromboxanes, lipoxygenase enzymes lead to leucotrienes (LT) and cytochrome P450 enzymes lead to hydroxyeicosatetraenoic and epoxyeicosatrienoic acids( Reference Lewis, Austen and Soberman 57 – Reference Kroetz and Zeldin 59 ). The decrease in arachidonic acid content of cell membrane phospholipids that occurs with incorporation of EPA and DHA (Fig. 1) reduces the availability of the usual eicosanoid substrate. Thus, increased incorporation of n-3 fatty acids into cell membranes is associated with decreased production of the major 2-series PG and 4-series LT( Reference Endres, Ghorbani and Kelley 26 – Reference Sperling, Benincaso and Knoell 29 , Reference Gibney and Hunter 30 – Reference Von Schacky, Kiefl and Jendraschak 42 ). This represents a key anti-inflammatory effect of n-3 fatty acids, and has been long recognised.

Fig. 2. Summary of eicosanoid synthesis from arachidonic acid. COX, cyclooxygenase; CYT P450, cytochrome P450 enzymes; DHET, dihydroxyeicosatrienoic acid; HETE, hydroxyeicosatetraenoic acid; HPETE, hydroperoxyeicosatetraenoic acid; EET, epoxyeicosatrienoic acid; LOX, lipoxygenase; LT, leucotriene; TX, thromboxane. Note that not all enzymes are named and that not all metabolites are shown.

EPA is also a substrate for the COX, lipoxygenase and cytochrome P450 enzymes, but the mediators produced have a different structure from those made from arachidonic acid (e.g. PGE3 rather than PGE2 and LTB5 rather than LTB4). Increased generation of 5-series LT has been demonstrated using macrophages from fish oil-fed mice( Reference Chapkin, Akoh and Miller 40 ) and neutrophils from human subjects taking fish oil supplements for several weeks( Reference Endres, Ghorbani and Kelley 26 , Reference Lee, Hoover and Williams 28 , Reference Sperling, Benincaso and Knoell 29 ). The functional significance of this is that the eicosanoids produced from EPA are often much less biologically active than those produced from arachidonic acid( Reference Goldman, Pickett and Goetzl 60 – Reference Bagga, Wang and Farias-Eisner 62 ). One reason for this reduced biological potency is that eicosanoid receptors typically have a much lower affinity for the EPA-derived mediator than for the arachidonic acid-derived one( Reference Wada, DeLong and Hong 63 ). Thus, EPA results in decreased production of potent eicosanoids from arachidonic acid and increased production of weak eicosanoids. Recently, the effect of arachidonic acid-derived PGD2 and EPA-derived PGD3 on neutrophil adhesive interactions with endothelial cells was investigated in an in vitro setting( Reference Tull, Yates and Maskrey 64 ). Both EPA and PGD3 were able to inhibit neutrophil transmigration through the endothelial cell monolayers, an effect which could be prevented by either arachidonic acid or PGD2. An antagonist to the PGD receptor DP1 also inhibited transmigration, while a DP1 agonist overcame the inhibitory effect of EPA. It was concluded that PGD2 acts to up-regulate neutrophil transmigration, analogous to neutrophilic infiltration into inflammatory sites, acting through DP1 while PGD3 acts to prevent this effect of PGD2 again acting at DP1. The observation that PGD3 can effectively compete with PGD2 is supported by the findings of Wada et al. ( Reference Wada, DeLong and Hong 63 ) that DP1 has a greater affinity for PGD3 than for PGD2.

New families of anti-inflammatory and inflammation resolving mediators are produced from EPA and DHA

Historically, research has focused on the initiation of inflammatory responses and strategies to promote or suppress. In the last 10 years or so a greater appreciation of the importance of ‘turning off’ inflammation has developed. This process is termed resolution and it seems likely that failure to resolve inflammation is an important factor in determining the course of inflammatory responses and their transition to disease states. During the recent past, lipid mediators produced from EPA and DHA have been discovered that seem to play a central role in resolution of inflammation. Hence, these mediators have been termed resolvins. Those produced from EPA are called E-series while those produced from DHA are termed D-series. Related compounds called protectins (also referred to as neuroprotectins when generated within neural tissue) are produced from DHA. The synthesis of resolvins and protectins involves the COX and lipoxygenase pathways operating across two cell types, with different epimers being produced in the presence and absence of aspirin( Reference Serhan, Clish and Brannon 65 – Reference Serhan, Chiang and van Dyke 68 ). Resolvin synthesis is increased by feeding fish-oil rich diets to laboratory rodents( Reference Hong, Gronert and Devchand 69 ) and was shown to occur in fat-1 mice in which colitis had been induced( Reference Hudert, Weylandt and Lu 70 ). A recent study revealed significant concentrations of resolvins E1 and D1 in the plasma of healthy human volunteers after supplementation with fish oil for 3 weeks( Reference Mas, Croft and Zahra 71 ).

The biological effects of resolvins and protectins have been examined extensively in cell culture and in animal models of inflammation, where they have been shown to be anti-inflammatory and inflammation resolving. For example, resolvins D1 and E1 and protectin D1 all inhibited transendothelial migration of neutrophils, preventing the infiltration of neutrophils into sites of inflammation; resolvin D1 inhibited IL-1β production; and protectin D1 inhibited TNF and IL-1β production( Reference Serhan, Clish and Brannon 65 – Reference Serhan, Chiang and van Dyke 68 ). Resolvins reduce inflammation and exert protection in experimental animals in models of inflammatory disease including arthritis( Reference Lima-Garcia, Dutra and da Silva 72 ), colitis( Reference Arita, Yoshida and Hong 73 ) and asthma( Reference Aoki, Hisada and Ishizuka 74 , Reference Haworth, Cernadas and Yang 75 ). The potent effects of resolvins and protectins may explain many of the anti-inflammatory effects of n-3 fatty acids.

n-3 Fatty acids inhibit activation of the pro-inflammatory transcription factor NF-κB

Cell culture studies with EPA and DHA show inhibition of LPS-induced production of COX-2, inducible nitric oxide synthase, TNF, IL-1, IL-6, IL-8 and IL-12 in endothelial cells( Reference De Caterina and Libby 76 , Reference Khalfoun, Thibault and Watier 77 ) and monocytes( Reference Lo, Chiu and Fu 78 , Reference Babcock, Novak and Ong 79 ). Animal feeding studies with fish oil, a source of EPA and DHA, support the observations made in vitro with respect to the effects of n-3 fatty acids on inflammatory cytokine production. For example, dietary fish oil decreased the production of TNF, IL-1β and IL-6 by LPS-stimulated mouse macrophages( Reference Yaqoob and Calder 39 , Reference Billiar, Bankey and Svingen 80 , Reference Renier, Skamene and de Sanctis 81 ). Some studies in healthy human subjects have demonstrated that oral fish oil supplements can decrease production of TNF, IL-1β, IL-6 and various growth factors by LPS-stimulated monocytes or mononuclear cells( Reference Endres, Ghorbani and Kelley 26 , Reference Caughey, Mantzioris and Gibson 27 , Reference Gibney and Hunter 30 , Reference Baumann, Hessel and Larass 82 – Reference Abbate, Gori and Martini 84 ), although not all studies confirm this effect.

NF-κB is a key transcription factor involved in up-regulation of inflammatory cytokine, adhesion molecule and COX-2 genes( Reference Kumar, Takada and Boriek 85 , Reference Sigal 86 ). Inactive NF-κB is a trimer localised within the cytosol; it is activated via a signalling cascade triggered by extracellular inflammatory stimuli which involves phosphorylation of an inhibitory subunit of NF-κB (IκB) which then dissociates allowing translocation of the remaining NF-κB dimer to the nucleus( Reference Perkins 87 ). LPS induces inflammation by activating NF-κB, as do some inflammatory cytokines and UV irradiation. EPA or fish oil has been shown to decrease LPS-induced activation of NF-κB in human monocytes( Reference Lo, Chiu and Fu 78 , Reference Novak, Babcock and Jho 88 , Reference Zhao, Joshi-Barve and Barve 89 ) and this was associated with decreased IκB phosphorylation( Reference Novak, Babcock and Jho 88 , Reference Zhao, Joshi-Barve and Barve 89 ).

Until fairly recently it has not been clear how n-3 fatty acids could influence NF-κB activation. However, recent studies suggest several possible mechanisms that might be involved. PPARγ is a transcription factor that acts in an anti-inflammatory manner( Reference Szanto and Nagy 90 ). It is able to directly regulate inflammatory gene expression, but it also interferes physically with the activation of NF-κB( Reference Van den Berghe, Vermeulen and Delerive 91 ). DHA induced PPARγ in dendritic cells and this was associated with inhibition of NF-κB activation and reduced production of the pro-inflammatory cytokines TNF and IL-6 following LPS stimulation( Reference Kong, Yen and Vassiliou 92 ). In addition, DHA induced a number of known PPARγ target genes in dendritic cells, suggesting this as an important anti-inflammatory mechanism of action of DHA and perhaps also of EPA( Reference Zapata-Gonzalez, Rueda and Petriz 93 ).

The n-3 fatty acid mediated inhibition of NF-κB activation is associated with decreased IκB phosphorylation( Reference Novak, Babcock and Jho 88 , Reference Lee, Sohn and Rhee 94 ). In contrast with the effects of EPA and DHA on IκB phosphorylation and subsequent activation of NF-κB, SFA, especially lauric acid, enhanced IκB phosphorylation and NF-κB activation in macrophages( Reference Lee, Sohn and Rhee 94 ) and dendritic cells( Reference Weatherill, Lee and Zhao 95 ) and so promoted inflammatory gene expression. Lee et al. ( Reference Lee, Sohn and Rhee 94 ) found that EPA and DHA were able to prevent the NF-κB mediated pro-inflammatory effect of lauric acid in macrophages. They also showed that the activation of NF-κB and induction of COX-2 expression by lauric acid did not occur in macrophages expressing a dominant-negative mutant of the cell surface LPS receptor, TLR-4, suggesting that lauric acid somehow interacts with TLR-4. Myeloid differentiation primary response gene 88 is a cell membrane-associated adapter protein used by TLR-4 to activate NF-κB. DHA inhibited COX-2 expression in macrophages bearing constitutively active TLR-4 but not in those bearing constitutively active myeloid differentiation primary response gene 88 suggesting that the effects of DHA are at the level of TLR-4( Reference Lee, Sohn and Rhee 94 ). More recently, Wong et al. ( Reference Wong, Kwon and Choi 96 ) demonstrated that exposure of macrophages to lauric acid-induced association of TLR-4, myeloid differentiation primary response gene 88 and other signalling proteins into membrane rafts in much the same way as LPS acts. Furthermore, they showed that DHA inhibited the ability of both LPS and lauric acid to promote recruitment of these signalling proteins into rafts. Thus, the differential effects of fatty acids on inflammatory signalling initiated through TLR-4 and impacting on NF-κB appear to relate to their ability to promote or disrupt raft formation within the membrane of inflammatory cells.

Activation of PPARγ and disruption of lipid rafts that initiate inflammatory signalling represent two mechanisms by which n-3 fatty acids could inhibit activation of NF-κB. Recently, a third mechanism has been identified( Reference Oh, Talukdar and Bae 97 ). This involves a cell surface G-protein coupled receptor called GPR120 which is highly expressed on inflammatory macrophages. The GPR120 agonist GW9508 inhibited responsiveness of macrophages to LPS( Reference Oh, Talukdar and Bae 97 ). The effect of GW9508 involved reduced phosphorylation of IκB kinase and IκB, IκB maintenance in the cytosol and reduced TNF and IL-6 production. These observations suggest that GPR120 signalling is anti-inflammatory. Both, EPA and DHA, but not arachidonic, palmitic or myristic acids, promoted GPR120-mediated gene activation, although they were less potent than GW9508. The effects of DHA were further explored( Reference Oh, Talukdar and Bae 97 ). Its inhibitory effects on LPS-induced IκB kinase phosphorylation, IκB phosphorylation and degradation, and TNF, IL-6 and monocyte chemotactic protein-1 production did not occur in GPR120 knockdown cells. These observations suggest that the inhibitory effect of DHA (and probably also those of EPA) on responsiveness to LPS occur via GPR120, which induces signalling that interferes with the pathway that activates NF-κB.

Thus, recent studies suggest three alternative mechanisms by which EPA and DHA might act to suppress inflammatory signalling via NF-κB: activation of PPARγ which physically interacts with NF-κB preventing its nuclear translocation, interfering with early membrane events involved in activation of NF-κB via TLR-4 and action via GPR120 which initiates an anti-inflammatory signalling cascade that inhibits signalling leading to NF-κB activation (Fig. 3). The extent to which these three mechanisms are interlinked is not clear at this stage.

Fig. 3. Summary of different mechanisms by which n-3 fatty acids inhibit activation of the pro-inflammatory transcription factor NF-κB. COX, cyclooxygenase; GPR, G-protein coupled receptor; PPAR, peroxisome proliferator activated receptor. Dotted lines indicate inhibition.

n-3 Fatty acids affect the formation of signalling platforms (rafts) in the plasma membrane of T-cells and in other immune cells

In cell cultures both EPA and DHA inhibit T-cell proliferation( Reference Calder, Yaqoob and Harvey 13 , Reference Calder, Bevan and Newsholme 16 , Reference Calder, Bond and Bevan 98 – Reference Calder and Newsholme 100 ) and the production of the key T-helper 1 type cytokine IL-2( Reference Calder, Yaqoob and Harvey 13 , Reference Calder and Newsholme 99 , Reference Calder and Newsholme 100 ). Animal feeding studies with fairly high amounts of fish oil, or of individual n-3 fatty acids, have also reported reduced T-cell proliferative responses( Reference Yaqoob, Newsholme and Calder 101 – Reference Fowler, McMurray and Fan 104 ) and alterations in T-helper 1 cytokine gene expression( Reference Wallace, Miles and Evans 102 ) and production( Reference Jolly, Jiang and Chapkin 103 ). Studies in human subjects are less consistent, although some studies have shown that increased intake of EPA+DHA decreases human T-cell proliferation( Reference Thies, Nebe-von-Caron and Powell 33 , Reference Meydani, Endres and Woods 38 ) and IL-2 production( Reference Meydani, Endres and Woods 38 ). These functional effects of n-3 fatty acids on T-cells have been linked with changes in membrane order( Reference Calder, Yaqoob and Harvey 13 ), altered patterns of eicosanoid production( Reference Calder, Bevan and Newsholme 16 ) and modification of early signal transduction events within the plasma membrane, including reduced generation of diacylglycerol( Reference Jolly, Jiang and Chapkin 103 , Reference Fowler, McMurray and Fan 104 ) and inhibition of the activation of specific isoforms of protein kinase C( Reference May, Southworth and Calder 105 , Reference Denys, Hichami and Khan 106 ) and of mitogen-activated protein kinases( Reference Denys, Hichami and Khan 107 , Reference Denys, Hichami and Khan 108 ). Until fairly recently, the earliest event reported to be affected by n-3 fatty acids following T-cell activation was the phosphorylation of the signalling enzyme phospholipase C-γ1 which was decreased by fish oil feeding in rats( Reference Sanderson and Calder 109 ). This latter effect was confirmed in a T-cell line exposed to EPA( Reference Stulnig, Berger and Sigmund 110 ) and was extended to demonstrate further upstream events of EPA on signalling proteins in T-cells( Reference Stulnig, Berger and Sigmund 110 – Reference Zeyda, Staffler and Horejsi 112 ). The earliest event affected by EPA was reported to be the inhibition of the anchoring of the protein called linker of activated T-cells into the plasma membrane( Reference Zeyda, Staffler and Horejsi 112 ). These in vitro studies identified that the effects of EPA on early signalling events in T-cells seem to involve the disruption of the formation of signalling platforms in the plasma membrane termed lipid rafts( Reference Stulnig, Berger and Sigmund 110 – Reference Zeyda and Stulnig 115 ).

Lipid rafts are regions of membranes with a distinct, characteristic structural composition( Reference Simons and Toomre 116 , Reference Pike 117 ). They are particularly rich in sphingolipids and cholesterol, and the side chains of the phospholipids are usually highly enriched in SFA compared with the surrounding non-raft regions of the membrane. As a result of the presence of cholesterol and SFA, lipid rafts are more ordered (‘less fluid’) than the surrounding (non-raft) portions of the membrane. Cytoplasmic proteins that are covalently modified by SFA (palmitoyl or myristoyl moieties) and cell surface proteins that are attached via a glycosyl phosphatidylinositol anchor are highly concentrated within lipid raft regions. Many proteins involved in signal transduction, such as Src family kinases, G proteins, growth factor receptors, mitogen-activated protein kinases and protein kinase C are predominantly found in lipid rafts, which appear to act as signalling platforms by bringing together (i.e. co-localising) various signalling components, facilitating their interaction. The importance of rafts has been well demonstrated with respect to T-lymphocyte responses to activation( Reference Katagiri, Kiyokawa and Fujimoto 118 – Reference Yaqoob 121 ) and research now suggests that raft disruption underlies the mechanism of action of n-3 fatty acids on T-cells( Reference Kim, Khan and McMurray 53 , Reference Stulnig and Zeyda 114 , Reference Zeyda and Stulnig 115 , Reference Yaqoob 121 ) and other immune cells( Reference Shaikh, Jolly and Chapkin 54 , Reference Calder 122 , Reference Calder 123 ). As indicated earlier, cell culture studies have demonstrated that exposure to EPA modifies raft formation in T-cells in a way that impairs the intracellular signalling mechanisms in these cells( Reference Stulnig, Berger and Sigmund 110 – Reference Zeyda and Stulnig 115 ). Feeding studies with n-3 fatty acids in mice confirm modifications of raft structure and function, linked to altered T-cell responses( Reference Fan, McMurray and Ly 124 , Reference Fan, Ly and Barhoumi 125 ).

In addition to n-3 fatty acids affecting lipid rafts in T-cells in ways that are linked to functional changes in the cells( Reference Stulnig, Berger and Sigmund 110 – Reference Zeyda and Stulnig 115 Reference Stulnig, Berger and Sigmund 110 – Reference Zeyda and Stulnig 115 , Reference Fan, McMurray and Ly 124 , Reference Fan, Ly and Barhoumi 125 ), effects on lipid rafts are also relevant to the influence of n-3 fatty acids on other cells of the immune system including inflammatory macrophages, dendritic cells and B-cells. In an earlier section it was described how Wong et al. ( Reference Wong, Kwon and Choi 96 ) had demonstrated that opposing effects of lauric acid and DHA and of LPS and DHA on responses of macrophages involved differential effects on lipid raft formation, ultimately linked to the NF-κB activation cascade. A series of studies has evaluated the in vitro effects of EPA and DHA and the effect of fish-oil feeding to mice on raft clustering in B-cells and the functional significance of the effects seen( Reference Shaikh and Edidin 126 – Reference Gurzell, Teague and Harris 131 ). An in vitro study using a B-cell line found that DHA, but not EPA, decreased raft clustering and that this was associated with increased movement of MHC I into rafts( Reference Shaikh, Rockett and Salameh 127 ), although these findings contrast with earlier observations that DHA decreased MHC I expression on the surface of B-cells due to an impairment of trafficking of MHC I from the endoplasmic reticulum to the Golgi and suppressed B-cell conjugation with T-cells( Reference Shaikh and Edidin 126 ). Feeding mice diets containing fish oil resulted in increased CD69 expression on the surface of stimulated splenic B-cells and increased the ex vivo production of inflammatory cytokines( Reference Rockett, Salameh and Carraway 128 – Reference Rockett, Teague and Harris 130 ), effects associated with impaired raft clustering and modification of raft size( Reference Rockett, Franklin and Harris 129 , Reference Rockett, Teague and Harris 130 ). Most recently Gurzell et al. ( Reference Gurzell, Teague and Harris 131 ) reported that feeding colitis-prone mice an n-3 fatty acid rich diet increased the EPA and DHA content of splenic B-cells, diminished the clustering of rafts in B-cells and resulted in larger membrane raft regions. These membrane effects were associated with enhanced B-cell activation ex vivo and increased inflammatory cytokine production. Thus, there are generally similar effects observed with a B-cell line in vitro ( Reference Shaikh, Rockett and Salameh 127 ) and with B-cells after feeding n-3 fatty acids to mice( Reference Rockett, Salameh and Carraway 128 – Reference Gurzell, Teague and Harris 131 ). The observations suggest that n-3 fatty acids enhance B-cell function and that this may be due to modulation of structure–function relationships within the plasma membrane. If this is correct then the effects of n-3 fatty acids on T-cells and B-cells might be different, although lipid raft disruption may be involved in both cases.

Summary and conclusions

Numerous effects of EPA and DHA on functional responses of cells involved in inflammation and immunity have been established over the last 40 years. These include inhibition of leucocyte chemotaxis, adhesion molecule expression and leucocyte-endothelial adhesive interactions, production of eicosanoids such as PG and LT from the n-6 fatty acid arachidonic acid, production of inflammatory cytokines like TNF and IL-6 and T-cell reactivity and enhanced phagocytosis (Table 1). These effects have been interpreted in the context of reducing inflammation that would lead to benefit in inflammatory conditions, as discussed elsewhere( Reference Calder 46 ). Fatty acid-induced modifications in membrane order and in the availability of substrates for eicosanoid synthesis are long-standing mechanisms that are considered important in explaining the anti-inflammatory and immunomodulatory actions of EPA and DHA. More recently, effects on signal transduction pathways and on gene expression profiles were identified to play a role. Over the last 10 years or so, significant advances in understanding the mechanisms of action of n-3 fatty acids have been made (Table 2). These include the identification of new actions of lipid mediators that were already described and of novel interactions among those mediators and the description of an entirely new family of lipid mediators, resolvins and protectins that have anti-inflammatory actions and, perhaps more importantly, are critical to the resolution of inflammation. These mediators may explain many of the existing actions of EPA and DHA. It is also recognised that EPA and DHA can inhibit activation of the prototypical inflammatory transcription factor NF-κB within classic inflammatory cells such as macrophages. Recent studies suggest three alternative mechanisms by which n-3 fatty acids might have this effect: activation of PPARγ which physically interacts with NF-κB preventing its nuclear translocation; interfering with early membrane events involved in activation of NF-κB via TLR-4; action via GPR120 which initiates an anti-inflammatory signalling cascade that inhibits signalling leading to NF-κB activation. Within T-cells, as well as other cells of relevance to immune and inflammatory responses, EPA and DHA act to disrupt very early events involving formation of the structures termed lipid rafts which bring together various proteins to form an effective signalling platform. The discovery of actions of EPA and DHA on lipid rafts and on the earliest signalling events in membranes and of a membrane receptor for n-3 fatty acids (GPR120) will re-focus attention on the membrane as the key site of action of these important bioactive fatty acids.

Table 2. Summary of the mechanisms of action of n-3 fatty acids (EPA+DHA) on immune and inflammatory cells

Acknowledgements

The author serves on Scientific Advisory Boards of the Danone Research Centre in Specialised Nutrition, Aker Biomarine, Pronova BioPharma and Smartfish; acts as a consultant to Mead Johnson Nutritionals, Vifor Pharma, Amarin Corporation and Qualitas; has received speaking honoraria from Solvay Healthcare, Solvay Pharmaceuticals, Pronova Biocare, Fresenius Kabi, B. Braun, Abbott Nutrition, Baxter Healthcare, Nestle, Unilever and DSM; and currently receives research funding from Vifor Pharma.

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Figure 0

Table 1. Summary of the effects of n-3 fatty acids (EPA+DHA) on immune and inflammatory cells

Figure 1

Fig. 1. Time course of incorporation of EPA and DHA and of disappearance of arachidonic acid in human mononuclear cells in healthy volunteers consuming fish oil. Healthy human volunteers consumed fish oil providing 2·1 g EPA and 1·1 g DHA per d for 1 week(56) or for 12 weeks(31). Blood was sampled at several time points in each study and mononuclear cells prepared. Fatty acid composition of the cells was determined by GC. Mean values are shown. ▪ and , EPA; • and , DHA; ▴ and , arachidonic acid; Black symbols represent data from Faber et al.(56); Grey symbols represent data from Yaqoob et al.(31).

Figure 2

Fig. 2. Summary of eicosanoid synthesis from arachidonic acid. COX, cyclooxygenase; CYT P450, cytochrome P450 enzymes; DHET, dihydroxyeicosatrienoic acid; HETE, hydroxyeicosatetraenoic acid; HPETE, hydroperoxyeicosatetraenoic acid; EET, epoxyeicosatrienoic acid; LOX, lipoxygenase; LT, leucotriene; TX, thromboxane. Note that not all enzymes are named and that not all metabolites are shown.

Figure 3

Fig. 3. Summary of different mechanisms by which n-3 fatty acids inhibit activation of the pro-inflammatory transcription factor NF-κB. COX, cyclooxygenase; GPR, G-protein coupled receptor; PPAR, peroxisome proliferator activated receptor. Dotted lines indicate inhibition.

Figure 4

Table 2. Summary of the mechanisms of action of n-3 fatty acids (EPA+DHA) on immune and inflammatory cells